The cloning of DNA segments is performed as a daily routine in many research labs. It is frequently performed in order to move a first polynucleotide sequence from a first vector into a second vector, where the second vector performs a function that is not performed by the first. Differences between the two vectors may include differences in selectable markers or differences in replicative sequences. They may also include differences in vector sequence elements that may directly interact with the first polynucleotide, for example by affecting expression of the first polynucleotide, or by encoding polypeptides that interact with or are joined to polypeptides encoded by the first polynucleotide.
The specialized vectors used for subcloning DNA segments are functionally diverse. These include but are not limited to: vectors for expressing genes in various organisms; for regulating gene expression; for providing tags to modify polypeptide properties such as solubility, localization, affinity for a substrate, color, fluorescence, characteristics that facilitate protein purification and characteristics that facilitate tracking of proteins in cells; for modifying the cloned DNA segment (e.g., generating deletions); for the synthesis of probes (e.g., riboprobes); for the preparation of templates for DNA sequencing; for the identification of protein coding regions; for the fusion of various protein-coding regions; for expressing one or more enzymes to catalyze a reaction and for providing large amounts of the DNA of interest. It is common that a particular investigation will involve subcloning the DNA segment of interest into several different specialized vectors.
A great deal of time can be expended in the cloning of DNA segments. The basic methods of cloning have been known for many years and have changed little during that time. A typical cloning protocol based on restriction enzyme digestion is as follows: (1) digest the DNA to be cloned with one or two restriction enzymes; (2) purify the digested DNA segment of interest to be cloned; (3) prepare the vector by cutting with appropriate restriction enzymes, treating with alkaline phosphatase, purifying as appropriate; (4) ligate the DNA segment to the vector, with appropriate controls to estimate background of uncut and self-ligated vector; and (5) introduce the resulting vector into an E. coli host cell; (6) pick selected colonies and grow small cultures overnight; (7) purify plasmid DNA; and (8) analyze the isolated plasmid on agarose gels (often after diagnostic restriction enzyme digestions) or by PCR.
As known in the art, simple subclonings can be done in one day (e.g., the DNA segment is not large and the restriction sites are compatible with those of the subcloning vector). However, many other subclonings can take several weeks, especially those involving unknown sequences, long fragments, toxic genes, unsuitable placement of restriction sites, high backgrounds or impure enzymes. Subcloning DNA fragments is thus often viewed as a chore to be done as few times as possible.
Accordingly, subcloning methods, using traditional restriction enzymes and ligase, are time consuming and relatively unreliable. There is thus a need for a rapid and reliable method for moving a polynucleotide into a plurality of specialized vectors. There is also a need for a rapid and reliable method for moving a plurality of polynucleotides into a single specialized vector.
Furthermore, site specific recombinases have been used to recombine DNA in vitro and in vivo. However, significant disadvantages of such methods include the time required to perform the reaction, the cost of the reagents, and the unavoidable incorporation of specific recombinase recognition sequences into the final construct: these sequence “scars” can interfere with the functions of other sequence elements within the construct.
Accordingly, there is a need in the art for an alternative rapid cloning system that provides advantages over the known use of multiple cloning sites or engineered recombination sites.